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U N I T 4
Infection and Immunity
Serology
Serology
—literally, “the study of serum”—is an indi-
rect means of identifying infectious agents by measur-
ing serum antibodies in the diseased host. A tentative
diagnosis can be made if the antibody level, also called
antibody titer
, against a specific pathogen rises during
the acute phase of the disease and falls during conva-
lescence. Serologic identification of an infectious agent
is not as accurate as culture, but it may be a useful
adjunct, especially for the diagnosis of diseases caused
by pathogens such as the hepatitis B virus that cannot
be cultured or diagnosis of past diseases. The measure-
ment of antibody titers has another advantage in that
specific antibody types such as IgM and IgG are pro-
duced by the host during different phases of an infec-
tious process. IgM-specific antibodies generally rise and
fall during the acute phase of the disease, whereas the
synthesis of the IgG class of antibodies increases during
the acute phase and remains elevated until or beyond
resolution.
Measurements of class-specific antibodies are also
useful in the diagnosis of congenital infections.
IgM antibodies do not cross the placenta, but cer-
tain IgG antibodies are transferred passively from
mother to child during the final trimester of gestation.
Consequently, an elevated level of pathogen-specific
IgM antibodies in the serum of a neonate must have
originated from the child and therefore indicates con-
genital infection. A similarly increased IgG titer in
the neonate does not differentiate congenital from
maternal infection.
The technology of
direct antigen detection
incorpo-
rates features of culture and serology but reduces to a
fraction the time required for diagnosis. In principle,
this method relies on purified antibodies to detect anti-
gens of infectious agents in specimens obtained from
the diseased host. Common sources of these antibod-
ies are
hybridomas
, cell lines created by fusing normal
antibody-producing spleen cells from an immunized ani-
mal with malignant myeloma cells. The resulting hybrid
synthesizes large quantities of so-called
monoclonal
antibodies
that are highly specific for a single antigen
and a single pathogen.
The antibodies are labeled with a substance that
allows microscopic or overt detection when bound
to the pathogen or its products. In general, the three
types of labels used for this purpose are fluorescent
dyes, enzymes, and particles such as latex beads.
Fluorescent antibodies allow visualization of an infec-
tious agent with the aid of fluorescence microscopy.
Depending on the type of fluorescent dye used, the
organism may appear bright green or orange against
a black background, making detection extremely easy.
Enzyme-labeled antibodies function in a similar man-
ner. The enzyme is capable of converting a colorless
compound into a colored substance, thereby permit-
ting detection of antibody bound to an infectious
agent without the use of a fluorescent microscope.
Particles coated with antibodies clump together, or
agglutinate, when the appropriate antigen is present in
a specimen. Particle agglutination is especially useful
when examining infected body fluids such as urine,
serum, or spinal fluid.
Protein Detection
Mass spectrometry is a technique for determining the
composition of a sample. It generates a protein-based
profile or “fingerprint” from microbes that is unique
to a given species. By analyzing the proteins that make
up bacteria, yeast, or molds, clinical laboratories can
quickly fingerprint these organisms and identify them
based on the size and number of proteins detected.
For example, analysis of bacteria such as
S. aureus
can often be accomplished by direct analysis of colony
growth by the mass spectrometer within minutes of
bacterial growth.
DNA and RNA Detection
Methods for identifying a pathogen by its unique DNA
or RNA sequence are increasingly being used. Several
techniques have been devised to accomplish this goal,
each having different degrees of sensitivity regarding the
number of organisms that need to be present in a speci-
men for detection.
The first of these methods is called
DNA probe
hybridization.
Small fragments of DNA are cut from
the genome of a specific pathogen and labeled with
compounds (photo-emitting chemicals or antigens)
that allow detection. The labeled DNA probes are
added to specimens from an infected host. If the patho-
gen is present, the probe attaches to the complementary
strand of DNA on the genome of the infectious agent,
permitting rapid diagnosis. The use of labeled probes
has allowed visualization of particular agents within
and around individual cells in histologic sections of
tissue.
A second and more sensitive method of DNA detec-
tion is the
polymerase chain reaction
(PCR). This
method allows technicians to tag a segment of patho-
gen DNA—if present in the patient sample—and then
multiply it to detectable levels. To perform the assay,
a specimen containing the suspect pathogen is heated
(Fig. 14-11). This causes the double-stranded DNA
in the specimen to separate into single strands. It is
then allowed to cool. Next, two short DNA sequences
(usually less than 25 nucleotides long) called
prim-
ers
are added to the specimen. These primers locate
and bind only to the complementary target DNA of
the pathogen in question. Then, a heat-stable DNA
polymerase—an enzyme that catalyzes the synthesis
of DNA—is added. It begins to replicate the DNA
from the point at which the primers attached, similar
to two trains approaching each other on separate but
converging tracks. After the initial cycle, DNA polym-
erization ceases at the point where the primers were
located, producing two new strands of DNA. The
specimen is heated again, and the process starts anew.
